Study of oxidation products of morphine and their reactivity by electrochemistry/(liquid chromatography/)mass spectrometry

Abstract

Morphine is a potent analgesic that is commonly used for the clinical management of chronic and acute pain. Several studies indicated that some adverse reactions of morphine might be in part related to oxidation processes during phase I metabolism, resulting in generation of reactive metabolites, which could bind to glutathione (GSH) or macromolecules. In this work we utilized electrochemistry/(liquid chromatography/)mass spectrometry for in vitro study of the oxidative transformation of morphine. Morphine was oxidized in a thin layer flow cell equipped with a glassy carbon working electrode controlled by Roxy™ potentiostat. Using a purely instrumental approach, eighteen oxidation products of morphine were identified based on the accurate mass measurements, chromatographic retention time on a reversed-phase column and the fragmentation patterns. Incubation of morphine oxidation products with GSH resulted in formation of five adducts, including isomeric forms. We also demonstrated that morphine oxidation products could also bind to thiol groups of larger molecules, particularly human serum albumin (HSA). Analysis of the tryptic digest of HSA, isolated from human plasma after incubation with morphine oxidative metabolites, confirmed that the protein was modified at the only free cysteine residue (Cys34) with dihydroxylation product of morphinone.